Recombinant human FOXJ1 protein binds DNA, forms higher-order oligomers, has gel-shifting domains and contains intrinsically disordered regions

Abstract

Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into E. coli as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and E. coli host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in E. coli BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (Mr) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the E. coli cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization via the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.